Myosin molecular motors play crucial, dynamic roles in most cellular processes, including contraction, movement, and shape change. A variety of diseases owe their origins to defects in the myosin family of molecular motors: defects in myosins give rise to deafness (VI, VII and others), neurological deficits (myosin V), and cell division defects that may be linked to cancer (nonmuscle myosin II). In addition, inherited familial cardiomyopathies affecting 1 out of 500 people result from missense mutations in various cardiac muscle proteins, with mutations in beta-cardiac myosin being one of the most common sources of this disease. Thus, our long-term goal of achieving a detailed understanding of how myosins transduce the chemical energy of ATP hydrolysis into mechanical motion will be very important in clinical settings (note, there are now phase II clinica trials of a small molecule activator of cardiac myosin for congestive heart failure; Malik et al., Cardiac Myosin Activation: A Potential Therapeutic Approach for Systolic Heart Failure. Science 331: 1439 (2011)). Comparative studies of several myosin family members, using an interdisciplinary approach that has included in vitro motility and laser trap assays, have revealed key features of how myosins work. There is now firm evidence supporting the lever arm hypothesis for the origin of directional movement. Nevertheless, many pivotal issues remain. For example, what is the sequence of events in the conversion of the chemical energy derived from ATP hydrolysis to a mechanical stroke? How are these altered in a processive motor, where each of the catalytic heads is subject to mechanical forces from intramolecular tension? An important component of the proposed project is technology development, which has been a hallmark of this grant since its inception in the 1980s, when we established the first quantitative in vitro motility assays and later adaptations of laser traps for the study of molecular motors. Along these lines we will characterize gating of nucleotide kinetics by myosins V and VI using newly developed methods for visualizing the binding and release of nucleotide from the front and rear heads of processively moving molecules. We will also develop tools for accurately measuring the location and orientation of single fluorescent probes, which will then allow us to explore the relationships between the chemistry at the active site and the dynamics of the lever arms of myosin. All of these new methods will have important applications for studying the myosin mutations that are involved in disease processes. In addition, they will reach far beyond the field of myosin and even molecular motors, and will prove important for the single molecule community at large.